Cholesterol is transported in the blood by lipid-containing particles known as lipoproteins. The most important of these lipoproteins are high density lipoprotein (HDL), low density lipoprotein (LDL), very low density lipoprotein (VLDL), and lipoprotein (a), (Lp(a)). Of these components, HDL cholesterol can be measured directly by precipitating all apoB-containing lipoproteins (VLDL, LDL and Lp(a)). VLDL is the only fasting lipoprotein with triglyceride as the major lipid component. There are usually four grams of triglycerides for every gram of cholesterol in VLDL. LDL has traditionally been estimated by subtraction of HDL and VLDL cholesterol from total cholesterol.
Recently it has been demonstrated that Lp(a) is an important cholesterol-containing blood constituent. Several recent case-control studies have shown that plasma Lp(a) is elevated in subjects with coronary artery disease and advanced atherosclerosis. Lp(a) plasma levels appear to be inherited and do not fluctuate with diet. However, Lp(a) plasma levels can be reduced with niacin and neomycin. Since treatment is possible, screening the general public as part of a total lipid profile determination is a practical approach to identifying and reducing this risk factor.
Lp(a) has never been measured as a cholesterolcontaining blood component. In the past, only protein components of the Lp(a) molecule were measured. Today, most researchers have adopted the practice of estimating the entire mass of Lp(a) by estimating its most unique cholesterol apolipoprotein, namely apo(a). Not only does Lp(a) contain cholesterol (approximately 25% of total mass), it also contains apoB.sub.100, apo(a) and small amounts of triglyceride and phospholipid. Apo(a) accounts for only 8%-12% of the total mass of Lp(a).
Heretofore, several diagnostic kits based on ELISA technology have been proposed for measuring Lp(a). ELISA tests require a monoclonal antibody to detect a unique feature of Lp(a), of which there are few. The only component of Lp(a) that is unique to Lp(a) are small regions within the apo(a) molecule. The apo(a) molecule in total is not very unique since it is composed of regions that have 78-95% homology with another plasma protein, namely plasminogen. The largest portion of the apo(a) molecule is composed of repeating sequences of approximately 20,000 daltons that have 78-84% homology with the plasminogen region called kringle IV. The most unique feature of apo(a) is its high carbohydrate content. However, carbohydrate is not very antigenic in nature and would not be expected to contribute much to the development of unique monoclonal antibodies. These features make the production of monoclonal antibodies which react specifically to apo(a), and not to plasminogen, very difficult.
It would therefore be useful to have a simple, reliable test for measuring Lp(a). Current methods for determining Lp(a)-associated cholesterol indirectly by first determining apo(a) concentrations have a margin of error of + or -5%. This is due, in part, to the fact that apo(a) exists in a number of isoforms which can vary in mass by up to 50%. The method disclosed herein is a direct Lp(a) cholesterol determination and has a margin or error of + or -1%.